Publications by authors named "Alessandro Spilotros"

11 Publications

  • Page 1 of 1

Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism.

J Mol Biol 2018 09 2;430(18 Pt B):3157-3169. Epub 2018 Aug 2.

Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden. Electronic address:

Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopH) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopH state has an elevated affinity for SycH and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.
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http://dx.doi.org/10.1016/j.jmb.2018.07.014DOI Listing
September 2018

Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control.

BMC Biol 2018 07 11;16(1):76. Epub 2018 Jul 11.

Department of Chemistry, King's College London, Britannia House, Trinity Street, London, SE1 1DB, UK.

Background: Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The C-terminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo.

Results: We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain.

Conclusion: Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region.
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http://dx.doi.org/10.1186/s12915-018-0542-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042327PMC
July 2018

Progress in small-angle scattering from biological solutions at high-brilliance synchrotrons.

IUCrJ 2017 Sep 8;4(Pt 5):518-528. Epub 2017 Aug 8.

European Molecular Biology Laboratory, EMBL Hamburg c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.

Small-angle X-ray scattering (SAXS) is an established technique that provides low-resolution structural information on macromolecular solutions. Recent decades have witnessed significant progress in both experimental facilities and in novel data-analysis approaches, making SAXS a mainstream method for structural biology. The technique is routinely applied to directly reconstruct low-resolution shapes of proteins and to generate atomistic models of macromolecular assemblies using hybrid approaches. Very importantly, SAXS is capable of yielding structural information on systems with size and conformational polydispersity, including highly flexible objects. In addition, utilizing high-flux synchrotron facilities, time-resolved SAXS allows analysis of kinetic processes over time ranges from microseconds to hours. Dedicated bioSAXS beamlines now offer fully automated data-collection and analysis pipelines, where analysis and modelling is conducted on the fly. This enables SAXS to be employed as a high-throughput method to rapidly screen various sample conditions and additives. The growing SAXS user community is supported by developments in data and model archiving and quality criteria. This review illustrates the latest developments in SAXS, in particular highlighting time-resolved applications aimed at flexible and evolving systems.
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http://dx.doi.org/10.1107/S2052252517008740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5619845PMC
September 2017

Proteins involved in sleep homeostasis: Biophysical characterization of INC and its partners.

Biochimie 2016 Dec 24;131:106-114. Epub 2016 Sep 24.

Institute of Biostructures and Bioimaging, C.N.R., Via Mezzocannone 16, 80134 Napoli, Italy. Electronic address:

The insomniac protein of Drosophila melanogaster (INC) has a crucial role in sleep homeostasis as flies lacking the inc gene exhibit strikingly reduced and poorly consolidated sleep. Nevertheless, in vitro characterizations of INC biophysical properties and partnerships have not been yet reported. Here we report the heterologous expression of the protein and its characterization using a number of different techniques. Present data indicate that INC is endowed with a remarkable stability, which results from the cooperation of the two protein domains. Moreover, we also demonstrated and quantified the ability of INC to recognize its potential partners Cul3 and dGRASP. Taking into account the molecular organization of the protein, these two partners may be anchored simultaneously. Although there is no evident relationship between the reported INC functions and dGRASP binding, our data suggest that INC may cooperate as ligase adaptor to dGRASP ubiquitination. SAXS data collected on the complex between INC and Cul3, which represent the first structural characterization of this type of assemblies, clearly highlight the highly dynamic nature of these complexes. This strongly suggests that the functional behavior of these proteins cannot be understood if dynamic effects are not considered. Finally, the strict analogy of the biochemical/biophysical properties of INC and of its human homolog KCTD5 may reliably indicate that this latter protein and/or the closely related proteins KCTD2/KCTD17 may play important roles in human sleep regulation.
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http://dx.doi.org/10.1016/j.biochi.2016.09.013DOI Listing
December 2016

Virus Matryoshka: A Bacteriophage Particle-Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus.

Small 2016 Nov 16;12(42):5862-5872. Epub 2016 Sep 16.

Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, IN, 47405, USA.

Immature human immunodeficiency virus type 1 (HIV-1) is approximately spherical, but is constructed from a hexagonal lattice of the Gag protein. As a hexagonal lattice is necessarily flat, the local symmetry cannot be maintained throughout the structure. This geometrical frustration presumably results in bending stress. In natural particles, the stress is relieved by incorporation of packing defects, but the magnitude of this stress and its significance for the particles is not known. In order to control this stress, we have now assembled the Gag protein on a quasi-spherical template derived from bacteriophage P22. This template is monodisperse in size and electron-transparent, enabling the use of cryo-electron microscopy in structural studies. These templated assemblies are far less polydisperse than any previously described virus-like particles (and, while constructed according to the same lattice as natural particles, contain almost no packing defects). This system gives us the ability to study the relationship between packing defects, curvature and elastic energy, and thermodynamic stability. As Gag is bound to the P22 template by single-stranded DNA, treatment of the particles with DNase enabled us to determine the intrinsic radius of curvature of a Gag lattice, unconstrained by DNA or a template. We found that this intrinsic radius is far larger than that of a virion or P22-templated particle. We conclude that Gag is under elastic strain in a particle; this has important implications for the kinetics of shell growth, the stability of the shell, and the type of defects it will assume as it grows.
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http://dx.doi.org/10.1002/smll.201601712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6810630PMC
November 2016

LabDisk for SAXS: a centrifugal microfluidic sample preparation platform for small-angle X-ray scattering.

Lab Chip 2016 Apr;16(7):1161-70

Hahn-Schickard - Georges-Koehler-Allee 103, 79110 Freiburg, Germany.

We present a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering (SAXS) on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) features automatic generation of 20 different measurement conditions from three input liquids and (2) requires only 2.5 μl of protein solution, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. Total hands on time for preparation of 120 different measurement conditions is less than 5 min. Read-out is performed on disk within the synchrotron beamline P12 at EMBL Hamburg (PETRA III, DESY). We demonstrate: (1) aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirm fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7-4.4% CV of protein concentration). We apply the LabDisk for SAXS for basic analysis methods, such as measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. The suitability of the LabDisk for SAXS for protein structure analysis under different environmental conditions is demonstrated for glucose isomerase under varying protein and NaCl concentrations. We show that the apparent radius of gyration of the negatively charged glucose isomerase decreases with increasing protein concentration at low salt concentration. At high salt concentration the radius of gyration (Rg) does not change with protein concentrations. Such experiments can be performed by a non-expert, since the LabDisk for SAXS does not require attachment of tubings or pumps and can be filled with regular pipettes. The new platform has the potential to introduce routine high-throughput SAXS screening of protein structures with minimal input volumes to the regular operation of synchrotron beamlines.
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http://dx.doi.org/10.1039/c5lc01580dDOI Listing
April 2016

Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY).

J Appl Crystallogr 2015 Apr 12;48(Pt 2):431-443. Epub 2015 Mar 12.

European Molecular Biology Laboratory, Hamburg Outstation , Notkestrasse 85, Hamburg, 22603, Germany.

A high-brilliance synchrotron P12 beamline of the EMBL located at the PETRA III storage ring (DESY, Hamburg) is dedicated to biological small-angle X-ray scattering (SAXS) and has been designed and optimized for scattering experiments on macromolecular solutions. Scatterless slits reduce the parasitic scattering, a custom-designed miniature active beamstop ensures accurate data normalization and the photon-counting PILATUS 2M detector enables the background-free detection of weak scattering signals. The high flux and small beam size allow for rapid experiments with exposure time down to 30-50 ms covering the resolution range from about 300 to 0.5 nm. P12 possesses a versatile and flexible sample environment system that caters for the diverse experimental needs required to study macromolecular solutions. These include an in-vacuum capillary mode for standard batch sample analyses with robotic sample delivery and for continuous-flow in-line sample purification and characterization, as well as an in-air capillary time-resolved stopped-flow setup. A novel microfluidic centrifugal mixing device (SAXS disc) is developed for a high-throughput screening mode using sub-microlitre sample volumes. Automation is a key feature of P12; it is controlled by a beamline meta server, which coordinates and schedules experiments from either standard or nonstandard operational setups. The integrated SASFLOW pipeline automatically checks for consistency, and processes and analyses the data, providing near real-time assessments of overall parameters and the generation of low-resolution models within minutes of data collection. These advances, combined with a remote access option, allow for rapid high-throughput analysis, as well as time-resolved and screening experiments for novice and expert biological SAXS users.
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http://dx.doi.org/10.1107/S160057671500254XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379436PMC
April 2015

Multicore iron oxide mesocrystals stabilized by a poly(phenylenepyridyl) dendron and dendrimer: role of the dendron/dendrimer self-assembly.

Langmuir 2014 Jul 8;30(28):8543-50. Epub 2014 Jul 8.

Department of Chemistry and ‡Department of Biology, Indiana University , Bloomington, Indiana 47405, United States.

We report the formation of multicore iron oxide mesocrystals using the thermal decomposition of iron acetyl acetonate in the presence of the multifunctional and rigid poly(phenylenepyridyl) dendron and dendrimer. We thoroughly analyze the influence of capping molecules of two different architectures and demonstrate for the first time that dendron/dendrimer self-assembly leads to multicore morphologies. Single-crystalline ordering in multicore NPs leads to cooperative magnetic behavior: mesocrystals exhibit ambient blocking temperatures, allowing subtle control over magnetic properties using a minor temperature change.
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http://dx.doi.org/10.1021/la502409rDOI Listing
July 2014

SAXS analysis of the tRNA-modifying enzyme complex MnmE/MnmG reveals a novel interaction mode and GTP-induced oligomerization.

Nucleic Acids Res 2014 May 14;42(9):5978-92. Epub 2014 Mar 14.

Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussel, Belgium Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium

Transfer ribonucleic acid (tRNA) modifications, especially at the wobble position, are crucial for proper and efficient protein translation. MnmE and MnmG form a protein complex that is implicated in the carboxymethylaminomethyl modification of wobble uridine (cmnm(5)U34) of certain tRNAs. MnmE is a G protein activated by dimerization (GAD), and active guanosine-5'-triphosphate (GTP) hydrolysis is required for the tRNA modification to occur. Although crystal structures of MnmE and MnmG are available, the structure of the MnmE/MnmG complex (MnmEG) and the nature of the nucleotide-induced conformational changes and their relevance for the tRNA modification reaction remain unknown. In this study, we mainly used small-angle X-ray scattering to characterize these conformational changes in solution and to unravel the mode of interaction between MnmE, MnmG and tRNA. In the nucleotide-free state MnmE and MnmG form an unanticipated asymmetric α2β2 complex. Unexpectedly, GTP binding promotes further oligomerization of the MnmEG complex leading to an α4β2 complex. The transition from the α2β2 to the α4β2 complex is fast, reversible and coupled to GTP binding and hydrolysis. We propose a model in which the nucleotide-induced changes in conformation and oligomerization of MnmEG form an integral part of the tRNA modification reaction cycle.
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http://dx.doi.org/10.1093/nar/gku213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027165PMC
May 2014

The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin.

Proc Natl Acad Sci U S A 2012 Sep 27;109(37):14894-9. Epub 2012 Aug 27.

Department of Physics, University of Palermo, Via Archirafi 36, I-90123 Palermo, Italy.

The acknowledged success of the Monod-Wyman-Changeux (MWC) allosteric model stems from its efficacy in accounting for the functional behavior of many complex proteins starting with hemoglobin (the paradigmatic case) and extending to channels and receptors. The kinetic aspects of the allosteric model, however, have been often neglected, with the exception of hemoglobin and a few other proteins where conformational relaxations can be triggered by a short and intense laser pulse, and monitored by time-resolved optical spectroscopy. Only recently the application of time-resolved wide-angle X-ray scattering (TR-WAXS), a direct structurally sensitive technique, unveiled the time scale of hemoglobin quaternary structural transition. In order to test the generality of the MWC kinetic model, we carried out a TR-WAXS investigation in parallel on adult human hemoglobin and on a recombinant protein (HbYQ) carrying two mutations at the active site [Leu(B10)Tyr and His(E7)Gln]. HbYQ seemed an ideal test because, although exhibiting allosteric properties, its kinetic and structural properties are different from adult human hemoglobin. The structural dynamics of HbYQ unveiled by TR-WAXS can be quantitatively accounted for by the MWC kinetic model. Interestingly, the main structural change associated with the R-T allosteric transition (i.e., the relative rotation and translation of the dimers) is approximately 10-fold slower in HbYQ, and the drop in the allosteric transition rate with ligand saturation is steeper. Our results extend the general validity of the MWC kinetic model and reveal peculiar thermodynamic properties of HbYQ. A possible structural interpretation of the characteristic kinetic behavior of HbYQ is also discussed.
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http://dx.doi.org/10.1073/pnas.1205809109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443182PMC
September 2012

Conformational substates of ferricytochrome c revealed by combined optical absorption and electronic circular dichroism spectroscopy at cryogenic temperature.

Biophys Chem 2010 Mar 6;147(1-2):8-12. Epub 2009 Dec 6.

Department of Physical and Astronomical Sciences, University of Palermo, Via Archirafi 36, I-90123 Palermo, Italy.

We have investigated the heterogeneity of the Fe(III)-Met80 linkage of horse heart ferricytochrome c by probing the 695nm charge transfer band with absorption and electronic circular dichroism (ECD) spectroscopy. In order to verify the connection between conformational substates of the Fe(III)-Met80 linkage and the 695nm band spectral heterogeneity, we have performed experiments as a function of pH (neutral and acidic) and temperature (room and 20K). At room temperature, the ECD spectrum is blue shifted with respect to the absorption one; the shift is more pronounced at acidic pH and is compatible with the presence of sub-bands. ECD measurements at 20K highlighted the heterogeneous nature of the 695nm band and provided direct experimental evidence for the presence of sub-bands. Indeed, while the absorption spectra remained deceivingly unstructured, the ECD spectra showed well resolved peaks and shoulders. A consistent fit of the 20K absorption and ECD spectra showed that five Gaussians (each centered at the same frequency in the absorption and ECD spectrum) are able to reproduce the observed lineshapes. A careful analysis of frequency shifts and intensity ratios of these sub-bands enabled us to identify at least three distinct sub-bands arising from taxonomic conformational substates of the Fe(III)-Met80 linkage. In view of the major influence of the Fe(III)-Met80 linkage on the redox potential of ferricytochrome c, we speculate that these spectrally distinguishable substates may have different functional roles.
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http://dx.doi.org/10.1016/j.bpc.2009.12.001DOI Listing
March 2010